Valsa mali secretes an effector protein VmEP1 to target a K homology domain‐containing protein for virulence in apple

Abstract The K homology (KH) repeat is an RNA‐binding motif that exists in various proteins, some of which participate in plant growth. However, the function of KH domain‐containing proteins in plant defence is still unclear. In this study, we found that a KH domain‐containing protein in apple (Malus domestica), HEN4‐like (MdKRBP4), is involved in the plant immune response. Silencing of MdKRBP4 compromised reactive oxygen species (ROS) production and enhanced the susceptibility of apple to Valsa mali, whereas transient overexpression of MdKRBP4 stimulated ROS accumulation in apple leaves, indicating that MdKRBP4 is a positive immune regulator. Additionally, MdKRBP4 was proven to interact with the VmEP1 effector secreted by V. mali, which led to decreased accumulation of MdKRBP4. Coexpression of MdKRBP4 with VmEP1 inhibited cell death and ROS production induced by MdKRBP4 in Nicotiana benthamiana. These results indicate that MdKRBP4 functions as a novel positive regulatory factor in plant immunity in M. domestica and is a virulence target of the V. mali effector VmEP1.

KH domain-containing proteins, such as SPL11-interacting pro-tein1 (SPIN1) (Vega-Sanchez et al., 2008), HUA ENHANCER4 (HEN4) (Cheng et al., 2003;Ortuno-Miquel et al., 2019), FLK (Lim et al., 2004), PEPPER (PEP) (Ripoll et al., 2006), KHZ1, and KHZ2 (Yan et al., 2017), have been shown to control flowering. Furthermore, some KH domain-containing proteins are crucial for the stress response, such as resistance against Fusarium oxysporum (Thatcher et al., 2015), tomato mosaic virus (Fujisaki & Ishikawa, 2008), and abiotic stress (Chen et al., 2013;Guan et al., 2013). However, the role of KH domain-containing proteins in manipulating development in plants has only been reported Arabidopsis thaliana. Moreover, only a few studies have correlated KH domain-containing proteins with the plant immune response. Only A. thaliana FLK is known to regulate resistance against pathogenic bacteria (Fabian et al., 2021;Lim et al., 2004). Whether other KH domain-containing proteins are involved in regulating host resistance is still unclear. Therefore, it is necessary to identify other KH domain-containing proteins and examine their possible involvement in the plant immune response.
The ascomycete Valsa mali, which causes apple (Malus domestica) Valsa canker, secretes effector protein 1 (VmEP1) to inhibit BAXinduced programmed cell death  and targets host pathogenesis-related protein 10 (PR10) . However, the mechanism by which VmEP1 manipulates plant immunity is not known. Previously, we found that VmEP1 targeted MdPR10  and other host proteins, such as KH domain-containing protein HEN4-like (MdKRBP4). In the present study, we discovered Our data collectively indicate that VmEP1 suppresses plant immunity by promoting MdKRBP4 degradation.

| MdKRBP4 interacts with VmEP1
In our previous study, the secretory protein VmEP1, with no known function, was shown to suppress plant defence by interrupting MdPR10-mediated resistance against V. mali .
In the present study, VmEP1-GFP was expressed in N. benthamiana leaves and the fusion proteins and associated proteins were purified with anti-GFP magnetic beads. To uncover the virulence mechanism of VmEP1 in V. mali, liquid chromatography-tandem mass spectrometry analysis of the purified VmEP1 and related proteins detected peptides of KH domain-containing proteins (Table 1), with no peptides after purification of green fluorescent protein (GFP) alone.
The yeast two-hybrid (Y2H) assay showed that VmEP1 targets a KH domain-containing protein ( Figure 1a,  Figure 1b). In addition, we tested the interaction between cYFP and nYFP empty vectors; yellow fluorescence was not detected (Figure 1d). Further analysis performed to validate the results of the BiFC assay revealed VmEP1 localization in the nucleus, cytoplasm, and plasma membrane (Figure S1a), and MdKRBP4 localization in the nucleus ( Figure S1b). These results suggest that VmEP1 interacts with MdKRBP4 in the nucleus.
Subsequently, VmEP1-HA was coexpressed with MdKRBP4-GFP in N. benthamiana leaves, using GFP as a control, and a coimmunoprecipitation (Co-IP) assay was performed using anti-GFP magnetic beads. Proteins in each sample were detected by western blot with anti-GFP and anti-HA antibodies. The results indicate that all genes were successfully expressed in N. benthamiana leaves (Figure 1c).
The immunoblotting assay showed that VmEP1-HA was present in the final GFP-MdKRBP4-precipitated immunocomplex (Figure 1c), indicating that MdKRBP4 interacts with VmEP1. Altogether, these results suggest that MdKRBP4 physically interacts with VmEP1.

| MdKRBP4 induces cell death in N. benthamiana leaves
ROS are a critical signal that triggers and activates cell death in plants (Jacobson, 1996;Petrov & Van Breusegem, 2012). Therefore, we investigated whether MdKRBP4 induces cell death in apple leaves. Although RT-qPCR analysis showed an up-regulation of the hypersensitive response-related genes MdHSRP203J (Pontier et al., 1998) and MdHIN1 (Takahashi et al., 2004)  as the internal reference. Apple leaves were infiltrated with MdKRBP4 or empty vector (EV) control for 3 days. Relative transcript levels of genes were normalized to MdEF-1α and calibrated to the levels of the EV control (set as 1) (mean ± standard deviation; n = 3; **p < 0.01, ***p < 0.001, ****p < 0.0001, Student's t test). These experiments were repeated three times with similar results. KH domain-containing proteins possess a conserved VIGXXGXXI motif (Burd & Dreyfuss, 1994). We obtained VIGXXGXXI-containing proteins from the apple genome database (ASM211411 v1). A total of 39 VIGXXGXXI-containing proteins including MdKRBP4 (XP_028954217.1) were identified ( Figure 3h). Phylogenetic tree analysis divided these proteins into six subgroups (Figure 3h). We randomly selected and cloned genes from each subgroup.  Figure 3h). In addition, MdKRBP4 overexpression resulted in the highest electrolyte leakage, indicating maximum cell death (Figure 3i).

F I G U R E 3 Overexpression of
These results imply that MdKRBP4 is essential for plant immunity.

| MdKRBP4 positively regulates apple resistance to V. mali
Subsequently, we constructed RNA interference vectors and transferred them to apple plants via Agrobacterium-mediated transformation to investigate whether MdKRBP4 positively regulates immunity.
After RT-qPCR evaluation, we obtained two silencing lines (SL5 and SL6) in which MdKRBP4 expression was less than 35% of that in wild-

| VmEP1 inhibits plant immunity induced by MdKRBP4
We further tested whether VmEP1 affects MdKRBP4-induced cell death and ROS accumulation. The result showed that VmEP1 atten- that MdSRLK3, a G-type lectin S-receptor-like protein kinase from apple, induces cell death in N. benthamiana (Yin, 2018). Therefore, we used the coexpression of MdSRLK3 and VmEP1 as a control. The results showed that MdSRLK3-induced cell death was not affected by VmEP1 (Figure 5a), indicating a specific effect of VmEP1 on MdKRBP4. Collectively, these results suggest that VmEP1 inhibits host immunity by targeting MdKRBP4 and attenuating the immune response triggered by MdKRBP4.

| MdKRBP4 is a virulence target of VmEP1
Furthermore, we verified whether MdKRBP4 is a virulence target of VmEP1. A VmEP1 deletion mutant (ΔVmEP1)  and WT V. mali were inoculated on WT apple leaves and SL5 leaves.
The average lesion diameter of WT V. mali was smaller than that of
MdPR10 expression and ROS accumulation, reducing apple resistance to V. mali (Figure 7d).  Figure S3). AtHEN4 and MdKRBP4 were within the same subgroup, and multiple sequence alignment of the HEN4 subgroup indicated that they had a KH domain ( Figure S4). Subsequently, we identified eight genes (Table S2)  Phytophthora infestans (Wu et al., 1995). ROS are toxic to plants.

| DISCUSS ION
Studies have revealed that a regulated increase in ROS benefits cell differentiation and proliferation (Schafer & Buettner, 2001), while an excess leads to a series of physiological changes, such as nucleic acid degradation, lipid peroxidation, and enzyme inactivation (Lamb & Dixon, 1997;Trachootham et al., 2009). For example, cells (Levine et al., 1994). Interestingly, in the present study we de- Biotic and abiotic stresses lead to ROS accumulation. Rapid production of ROS is a typical characteristic of the hypersensitive response following the recognition of pathogen infection (Lamb & Dixon, 1997;Wojtaszek, 1997). Pathogens adopt measures to inhibit ROS production. For example, the Puccinia striiformis effector Pst18363 can target and stabilize wheat Nudix hydrolase 23 (TaNUDX23) to suppress ROS accumulation and facilitate infection (Yang et al., 2020). In the present study we detected ROS production in MdKRBP4-expressing leaves of apple ( Figure 2)  to facilitate infection (Lin et al., 2021). These studies illustrated that effectors control host immunity in various ways, especially manipulating host protein ubiquitination. In the present study we found that the proteasome inhibitor MG132 stabilized MdKRBP4 (Figure 7a), suggesting that the MdKRBP4 protein is degraded via the 26S proteasome pathway. We demonstrated that VmEP1 targets MdKRBP4 The infiltration of apple leaves was carried out as previously described . Briefly, Agrobacterium suspensions were used to prepare 50 ml Agrobacterium containing the pCAMBIA1302 constructs, which was then vacuum-infiltrated into apple seedling leaves under 100 kPa for 10 min. The treated seedlings were cultured on MS medium for 2 days and then used for experiments.

| Y2H assay
MdKRBP4 and VmEP1 were cloned into the binary vectors pGADT7 and pGBDT7, respectively. Primers used in this study are summarized in Table S3. The polyethylene glycol-mediated conversion method stated in the Yeast Protocols Handbook (Clontech) was used to transform binary vectors into Saccharomyces cerevisiae AH109. pGADT7-MdKRBP4 and pGBDT7-VmEP1 were cotransformed into yeast cells, and then transformants were selected on synthetic dropout (SD) medium without tryptophan (Trp) and leucine (Leu). Then, single clones were transferred onto SD medium lacking adenine (Ade), histidine (His), Leu, and Trp, containing X-α-Gal for selection of interaction.

| BiFC assay
MdKRBP4 and VmEP1 were cloned into the binary vectors cYFP and nYFP, respectively.

| Co-IP assay
Co-IP assays were executed to verify the protein interactions in vivo.
To construct the MdKRBP4-GFP vector, MdKRBP4 was cloned into pCAMBIA1302 via homologous recombination, resulting in pCAM-BIA1302-MdKRBP4, in which the expression of the VmEP1-GFP fusion is driven by a CaMV 35S promoter. VmEP1 was fused into the vector PICH86988 with an HA tag at its C-terminus. Table S3  The protein extracts were cleaned by two rounds of centrifugation at 15,000 × g for 10 min to remove tissue debris. Next, the total proteins were incubated with 20 μl anti-GFP magnetic beads (Epizyme) for 3 h at 4°C. Then, the magnetic beads were washed three times with TBST buffer (50 mM Tris-HCl, pH 7.5, 8 g/L NaCl, 0.2 g/L polysorbate 20) to elute the proteins, which were boiled in 1× SDS-PAGE loading buffer for 10 min. The proteins were separated by 10% SDS-PAGE and examined by western blot with anti-GFP antibody (Abmart, M20004, 1:5000 dilution) or anti-HA antibody (Abmart, M20003, 1:3000 dilution). Antibody binding was detected using chemiluminescent horseradish peroxidase substrate (Millipore). GFP IP experiments were carried out with an anti-GFP magnetic beads kit (Epizyme) following the manufacturer's instructions.

| IP and mass spectrometry analysis
For IP and mass spectrometry, the sequence of VmEP1 was inserted into the pCAMBIA1302 vector fused with GFP. Table S3 contains the information of primers used in this study. The total proteins of leaves expressing VmEP1 were extracted and purified in native lysis buffer.
The total protein mixture was centrifuged at 14,000 × g for 10 min and tissue debris was discarded. Next, 30 μl anti-GFP magnetic beads (Shanghai Epizyme Biomedical Technology) was mixed into the supernatant and samples were incubated for 3 h at 4°C on a rotator.
The anti-GFP magnetic beads were washed about five times using TBST and the beads were boiled with 1× SDS-PAGE loading buffer for 10 min. The total proteins were separated by 10% SDS-PAGE. The gel was then stained using a kit from Thermo (MAN0011539) following the manufacturer's instructions. After staining with silver stain and subsequent destaining, gel sections were excised and then digested with trypsin. Finally, mass spectrometry was conducted to identify interacting proteins as previously described (Hu et al., 2014).

| Construction of transgenic apple
RNA interference vectors for apple were generated using the plant expression vector pK7GWIWG2D. The specific PCR fragments of MdKRBP4 were inserted into pK7GWIWG2D(II). Table S3 contains the information of primers applied in the present study. Agrobacterium-mediated transformation was used for transforming apples (Dai et al., 2013). Briefly, 50 ml Agrobacterium suspension was prepared in which leaf segments were shaken gently for 5-8 min. Excess fluid was wiped off with sterile filter paper. These leaf segments were then cocultivated on MS agar containing 2 mg/ml thidiazuron (TDZ), 0.5 mg/ml indole-3-butyric acid (IBA), and 100 μM acetosyringone in dark conditions for 2 days. Immediately after cocultivation, explants were transferred to MS agar with 2 mg/L TDZ,

| Isolation of RNA and RT-qPCR
RNA was isolated from samples (50 mg) using the EasyPure Plant RNA kit (Transgen) and processed with DNase I. RNA samples (2 μg) were reverse-transcribed using high-capacity cDNA reverse transcription kits (Applied Biosystems) and subjected to qPCR using gene-specific primers (available upon request). cDNA samples were amplified using 2× RealStar Green Power mixture (GenStar) and a Roche LightCycler 96 SW1.1 real-time PCR system (Roche). The elongation factor 1α (EF-1α) gene of M. domestica and Actin of N. benthamiana were used for normalization, and gene expression was calculated by the comparative C t method. Primers used in this study are summarized in Table S3. All experiments were repeated independently three times.

| DAB staining
H 2 O 2 accumulation in plant tissue was examined by staining with DAB as described previously (Xiao et al., 2003). Leaf pieces were infiltrated in DAB solution (Sigma) (1 mg/ml, pH 3.8) and shaken at room temperature for 12 h in the light. The leaf pieces were then destained with 95% ethanol. The cleared leaves were transferred to 50% glycerol. A microscope (Olympus) was used to take photographs. The accumulation of ROS was evaluated by ImageJ.

| Trypan blue staining
N. benthamiana leaves expressing MdKRBP4 or empty vector were stained by boiling for 10 min in lactophenol-trypan blue solution (10 ml lactic acid, 10 ml glycerol, 10 g phenol, 10 mg trypan blue, 10 ml distilled water). Then, they were decoloured with gentle shaking in a chloral hydrate solution (2.5 g/ml) for 12 h. Samples were photographed under natural light.

| Electrolyte leakage
Cell death was quantified by determining electrolyte leakage using a previously described method (Ma et al., 2021;Nayyar & Chander, 2004). Samples from N. benthamiana (diameter 1 cm) expressing MdKRBP4 were immersed in nanopure water (5 ml) for 3 h at room temperature to determine the electrical conductivity (E 1 ). A conductivity meter (Five Easy Plus Conductivity) was used to measure the conductivity. Then the samples were boiled for 10 min and the second electrical conductivity (E 2 ) was measured after the fluid recovered to ambient temperature. Electrolyte leakage was calculated as follows: electrolyte leakage (%) = (E 1 /E 2 ) × 100. This assay was repeated three times.

CO N FLI C T O F I NTE R E S T
The authors of this paper declare no competing interests exist.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used in this study are available from the corresponding author.